1 CHARGE TO PANEL
Introduction The Food Quality Protection Act (FQPA) of 1996 requires that EPA

“ d evelop a screening program, using appropriate validated test systems and other scientifically relevant information, to determine whether certain substances may have an effect in humans that is similar to an effect produced by a naturally occurring estrogen, or other such endocrine effect as the Administrator may designate. ” Subsequent to passage of the FQPA the EPA formed a federal advisory committee, the
Endoc rine Disruptor Screening and Testing Advisory Committee (EDSTAC), to provide recommendations on how to implement its Endocrine Disruptor Screening Program (EDSP). The EDSP is described in detail at the following website: http://www.epa.gov/scipoly/oscpendo/

. Based on the recommendations from the EDSTAC (EDSTAC 1998), the EPA, in addition to the estrogen pathway, included the androgen and thyroid pathways of the endocrine system in
humans as well as in wildl ife. The EDSTAC also recommended the Agency adopt a two

- tiered screening and testing program. Tier 1 is an integrated battery of relatively short

- term in vitro and in vivo assays designed to detect the potential of a chemical to interact with endocrine s ystem, principally the estrogen, androgen, and thyroid hormonal pathways. Test chemicals determined to have the potential to interact with the endocrine system, based on a weight

- of

- evidence analysis of the results of Tier 1 screening and inclusive of othe r scientifically relevant information, would be considered for Tier 2 testing. Tier 2 has the purpose to “ characterize the nature, likelihood, and dose

- response relationship of endocrine disruption of estrogen, androgen, and thyroid in humans and wildlife

. ” Tier 2 tests consist of more comprehensive, long

- term tests during various life stages and multiple generations enhanced with endocrine

- specific endpoints across multiple taxonomic groups, including mammals, birds, fish, amphibians, and invertebrates. T he purpose of Tier 2 testing is to identify any potential adverse outcome and provide quantitative concentration

response information that may be used for risk assessment. The following ecotoxicity tests, as generally recommended by EDSTAC, are being cons idered as part of the EDSP Tier 2:

• Japanese Quail Two

- generation Test (JQTT)

• Medaka Multi

- generation Test (MMT)

• Larval Amphibian Growth and Development Assay (LAGDA)

• Mysid Two

- generation Toxicity Test (MTTT)

The Integrated Summary Reports (ISRs) and accom panying appendices for each assay provide a
detailed account of the historical development and validation of a standardized protocol for each Tier 2 ecotoxicity assay. The EPA is requesting the SAP to provide independent peer review of
each Tier 2 ecotoxic ity test method.

( The criteria used to guide the validation process for each test method were based on the

principles of validation developed by the U.S. Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) and the Organisation for Economic and Cooperative Development (OECD). These questions follow a format consistent with those
international validation standards and are s imilar to the questions provided for the EDSP T ier
I validation).2 Validation Charge Questions:

Japanese Quail Two

- Generation Test (JQTT)

1. A rationale for the test method should be available, including a clear statement of scientific

basis and the regulatory purpose and need for the test method. The EDSTAC described the Tier 2 tests as having the purpose to “ characterize the nature, likelihood, and dose

- response relationship of endocrine disruption of estrogen, androgen, and thyroid in humans and wildlife .” T ier 2 tests were designed to be definitive tests that gene rate sufficient data to characterize the specific hazard of the substance and provide sufficient information on dose

response and adverse effects to permit risk decisions.

Please comment on the rationale and purpose of the assay as part of the Tier 2 tes ting in the EDSP, as described in Sections 2.5 and 2.6 of the JQTT ISR

.

2. Test methods and their associated endpoint(s) should be scientifically relevant to the

biological processes of interest and should be demonstrated to be responsive to the specifi c type of effect/ toxicity of interest. Each species presents unique characteristics from a biological perspective and allows for specialized endpoints to address a specific toxicological
mode of action.

Please comment on the biological and toxicological relevan ce of the assay in regards to the stated purpose of characterizing endocrine disruptors, as described in Sections 3 and 4 of the JQTT ISR.

3. The test protocol should be sufficiently detailed and should include a description of what is

measured and how it is measured. The selection of endpoints within the assay should be reflective of the biological processes of interest and the endpoints should be intrinsically relevant and have established sensitivity. The test protocol should de monstrate the ability to measure the endpoints and provide adequate performance criteria for evaluation.

3.1 Please comment on the selection, optimization and demonstration of the assay endpoints, as outlined in Sections 4 and 5 of the JQTT ISR

.

Patho logical evaluation of histological tissue preparations is an established, sensitive and integral endpoint in the assessment of effects in long term in vivo assays. The tissues targeted for histopathological evaluation should be shown to be sensitive to ex posure and relevant to a mode of action or pathway determination. T issue samples from several organs or glands ( e.g. , kidney, liver, thyroid, gonads) underwent histopathological examination in the JQTT inter

lab oratory validation and a discussion of histopathology as an endpoint is provided in Section 4.1.13 of the JQTT ISR

. 3.2 Please comment on the value of histopathological analys e s in the JQTT assay for each of the tissue types examined and what (if any) critical information is gained from their inclusion or would be lost if histology were not examined.3 4. Demonstration of the test method performance should be based on the testing of reference

chemicals representative of the types of substances for which the test method wi ll be used. Test substances should adequately represent an appropriate range of responses and physical/chemical properties for which the test method is proposed to be appropriate. The selection of the most appropriate statistical approaches depends in pa rt on the nature of the data and also on the design of the validation study. Statistical and non

- statistical methods used to analyze should be described.

Please comment on the selection of test substances and methods (analytical and statistical where ap propriate) chosen for the demonstration and validation of the JQTT assay.

5. Considering the variability inherent in biological and chemical test methods, a test method needs to be repeatable and reproducible. A test is robust and reliable if the result s are repeatable and reproducible within a laboratory and between different laboratories, respectively. A test protocol should provide sufficient guidance to ensure proper and consistent performance
across labs and chemicals.

Please comment on the test method robustness and reliability and the repeatability and reproducibility of the results obtained with th e JQTT assay.

6. T he test protocol should be descriptive enough to be fully transferable to a competent

laboratory. The protocol should describe the methodology of the assay in a clear and concise
manner so that a laboratory could comprehend the objective

, conduct the assay

, observe and measure prescribed endpoints

, compile and prepare data for statistical analyses

, and report the
results

. Section 6 of the JQTT ISR outlines the process of and challenges experienced in the
inter

- laboratory studies.

Please comment on the transferability across labs and provide any suggestions or
recommendations for improvement of the JQTT assay

.

7 . The purpose of the validation process is to determine the readiness of a test for inclusion in a

testing program. A component of readiness of a test is the evaluation of the usefulness and
limitations of the test , including the classes and types of test substances that can and cannot be
tested.

Please comment on the strengths and/or limitations of the JQTT assay , as described in Section 7 of the JQTT ISR

.

8

. There is sufficient evidence to indicate that EDCs can disrupt normal development and

repro ductive success, however the sensitivity of the F 2 generation compared to the P 0 or F 1 is less clearly defined. In the JQTT, there is an increase in endpoint CVs with each subsequent generation (JQTT ISR Table 6

- 4), which indicates decreased power of disc rimination.4 Please comment from a scientific and risk assessment perspective on the value added of multiple generations in the JQTT assay

.5 Medaka Multigeneration Test (MMT)

Section 5 of the MMT ISR outlines the EPA proposed M edaka R eproduction T est (MRT) assay

which utilizes the culmination of knowledge and experience gained from many successive U.S. and Japanese studies over multiple years

.

1. A rationale for the test method should be available, including a clear statement of scientific

basis an d the regulatory purpose and need for the test method. The EDSTAC introduced the Tier 2 tests as having the purpose to “ characterize the nature, likelihood, and dose

- response relationship of endocrine disruption of estrogen, androgen, and thyroid in human s and wildlife .” T ier 2 tests were designed to be definitive tests which generate sufficient data to characterize the specific hazard of the substance and provide sufficient information on dose

response and adverse effects to permit risk decisions.

Please comment on the rationale and purpose of the assay as part of the Tier 2 testing in
the EDSP
, as described in Sections 2.1 and 2.2 of the MMT ISR.

2. Test methods and their associated endpoint(s) should be scientifically relevant to the

biological processes of interest and should be demonstrated to be responsive to the specific type of effect/toxicity of interest. Each species presents unique characteristics from a biological perspective and allows for specialized endpoints to address a specific t oxicological
mode of action.

Pl ease comment on the biological and toxicological relevan ce of the assay in regards to the stated purpose of characterizing endocrine disruptors, as described in Section 3 of the
MM T.

3. The test protocol should be suffi ciently detailed and should include a description of what is

measured and how it is measured. The selection of endpoints within the assay should be reflective of the biological processes of interest and the endpoints should be intrinsically
relevant and h ave established sensitivity. The test protocol should demonstrate the ability to measure the endpoints and provide adequate performance criteria for evaluation.

Please comment on the selection, optimization and demonstration of the assay endpoints,
as outlined in Section 3 of the MM T ISR

.

4. Demonstration of the test method performance should be based on the testing of reference

chemicals representative of the types of substances for which the test method will be used. Test substances should adeq uately represent an appropriate range of responses and physical/chemical properties for which the test method is proposed to be appropriate. The selection of the most appropriate statistical approaches depends in part on the nature of the data
and also on the design of the validation study. Statistical and non

- statistical methods used to analyze should be described.6 Please comment on the selection of test substances and methods (analytical and statistical where appropriate) chosen for the demonstration and validation of the MMT and MRT

assay s

.

5. Considering the variability inherent in biological and chemical test methods, a test method needs to be repeatable and reproducible. A test is robust and reliable if the results are repeatable and reproducibl e within a laboratory and between different laboratories, respectively. A test protocol should provide sufficient guidance to ensure proper and consistent performance
across labs and chemicals.

Please comment on the test method robustness and reliabili ty and the repeatability and reproducibility of the results obtained with th e MTT and MRT assay s

.

6. T he test protocol should be descriptive enough to be fully transferable to a functional

laboratory. The protocol should describe the methodology of the a ssay in a clear and concise manner so that a laboratory could comprehend the objective, conduct the assay, observe and measure prescribed endpoints, compile and prepare data for statistical analyses, and report the
results. Section 4 of the MM T ISR outlin es the process of and challenges experienced in the
inter

- laboratory studies and Section 5 presents the optimization and proposed Medaka Reproduction Test (MRT) protocol

. Please comment on the transferability across labs and provide any suggestions or
recommendations for improvement of the

. MMT and MRT assays

.

7. The purpose of the validation process is to determine the readiness of a test for inclusion in a

testing program. A component of readiness of a test is the evaluation of the usefulness and
lim itations of the test , including the classes and types of test substances that can and cannot be
tested.

Please comment on the strengths and/or limitations of the MMT and MRT assay s

.

8. There is sufficient evidence to indicate that EDCs can disrupt no rmal development and

reproductive success, however the value added of the F 2 generation compared to the P 0 or F 1 is less clearly defined. The EPA is proposing the Medaka Reproduction Test (MRT) as an EDSP
Tier 2 assay. Th e MRT test terminates after the F 2 embryo hatch

, and a rationale for the proposed protocol is provided in Section 5 of the MMT ISR

. Please comment on the Agency’s rationale that the value added by the F2 generation is not sufficient to warrant its inclusion in the Tier 2 fish test protocol.

9. It is the Agency’s opinion that the outcomes of the various MMT trials have provided enough

information t o recommend a medaka reproduction test (MRT) for use as the fish test in Tier 2 of the EDSP. Two major changes from the MMT are proposed, i.e., an increase in the number of replicates per treatment for evaluating effects on reproduction, and terminating th e test after7 the embryos hatch in F 2 . Other proposed changes include minimizing the collection of endpoint
data from F 0 , and evaluating pathology in only the F 1 adults sampled after the assessment of reproduction.

Overall, the authors conclude that both the MMT and the MRT are transferable methods and are capable of adequately characterizing potential disruption of the endocrine system by putative e ndocrine disrupting chemicals. However, the MRT is recommended as the preferred
EDSP Tier 2 test method for fish because it is less resource intensive with improved statistical

power, app ears to be as sensitive, and is better able to ensure consistent findings when performed routinely by testing laboratories.

Please comment on the scientific rationale of the A gency’s proposed Tier 2 fish test, the MRT, with respect to statistical power, sensitivity, and consistency in performance across laboratories. In addition, please comment on the adequacy of the MRT to characterize potential endocrine disruption
, a require ment of Tier 2 of the EDSP.8 Larval Amphibian Growth and Development Assay (LAGDA)

1. A rationale for the test method should be available, including a clear statement of scientific

basis and the regulatory purpose and need for the test method. The EDSTAC introduced the Tier 2 tests as having the purpose to “ characterize the nature, likelihood, and dose

- response relationship of endocrine disruption of estrogen, androgen, and thyroid in humans and wildlife .” Tier 2 tests were designed to be definitiv e tests which generate sufficient data to characterize the specific hazard of the substance and provide sufficient information on dose

response and adverse effects to permit risk decisions.

Please comment on the rationale and purpose of the assay as part of the Tier 2 testing in the EDSP, as described in Section 3 of the LAGDA ISR.

2. Test methods and their associated endpoint(s) should be scientifically relevant to the

biological processes of interest and should be demonstrated to be responsive to the specific type of effect/toxicity of interest. Each species presents unique characteristics from a biological perspective and allows for specialized endpoints to address a specific toxicological
mode of action.

Please comment on the biological and toxi cological relevan ce of the assay in regards to the stated purpose of characterizing endocrine disruptors, as described in Sections 3 and
4 of the LAGDA ISR.

3. The test protocol should be sufficiently detailed and should include a description of what is

measured and how it is measured. The selection of endpoints within the assay should be reflective of the biological processes of interest and the endpoints should be intrinsically relevant and have established sensitivity. The test protocol should demon strate the ability to measure the endpoints and provide adequate performance criteria for evaluation.

3.1 Please comment on the selection, optimization and demonstration of the assay endpoints, as outlined in Sections 3 and 4 of the LAGDA ISR

.

Three of the four labs in the inter

- laboratory validation of the LAGDA were unsuccessful in properly performing the thyroxine ( T4 ) analyses using commercially available enzyme

- linked Immunosorbent assay (ELISA) kits with antibodies specific for human or canine T 4

. Subsequently, the EPA developed an extraction method (Section 5.3.2) and presents a revised
ELISA method for measuring T4 in amphibian samples in Appendix 7 of the LAGDA ISR

. 3.2 Please comment on the technical feasibility, reproducibility
, and accur acy of the
revised ELISA T4 measurement method. Provide any recommendations regarding additional guidance to ensure the reproducibility of T4 measurements.9 Vitellogenin (VTG) is an established biomarker of estrogenic exposure and is used as a key endpoint in endocrine disruptor testing. There is currently not a standardized commercial
source for Xenopus laevis VTG antibodies for an ELISA.

3.3 Please comment on the protocol for measur ing and reporting VTG levels . P rovide any recommendations rega rding additional guidance to ensure the consistency, repeatability and reproducibility of VTG measurements.

4. Demonstration of the test method performance should be based on the testing of reference

chemicals representative of the types of substances f or which the test method will be used. Test substances should adequately represent an appropriate range of responses and physical/chemical properties for which the test method is proposed to be appropriate. The selection of the most appropriate statistic al approaches depends in part on the nature of the data and also on the design of the validation study. Statistical and non

- statistical methods used to analyze should be described.

Please comment on the selection of test substances and methods (analytic al and statistical where appropriate) chosen for the demonstration and validation of the LAGDA assay.

5. Considering the variability inherent in biological and chemical test methods, a test method needs to be repeatable and reproducible. A test is robus t and reliable if the results are repeatable and reproducible within a laboratory and between different laboratories, respectively. A test protocol should provide sufficient guidance to ensure proper and consistent performance
across labs and chemicals.

Please comment on the test method robustness and reliability and the repeatability and reproducibility of the results obtained with th e LAGDA assay

.

6. T he test protocol should be descriptive enough to be fully transferable to a functional

laboratory. The protocol should describe the methodology of the assay in a clear and concise manner so that a laboratory could comprehend the objective, conduct the assay, observe and measure prescribed endpoints, compile and prepare data for statistical analyses, a nd report the results. Sections 5 and 6 of the LAGDA ISR outline the process of and challenges experienced
in the inter

- laboratory studies.

Please comment on the transferability across labs and provide any suggestions or
recommendations for improvement of the LAGDA assay

.

7. The purpose of the validation process is to determine the readiness of a test for inclusion in a

testing program. A component of readiness of a test is the evaluation of the usefulness and
limitations of the test , including the cla sses and types of test substances that can and cannot be
tested.

Please comment on the strengths and/or limitations of the LAGDA assay.10 Mysid Two

- Generation Toxicity Test (MTTT)

1. A rationale for the test method should be available, including a cle ar statement of scientific

basis and the regulatory purpose and need for the test method. The EDSTAC introduced the Tier 2 tests as having the purpose to “ characterize the nature, likelihood, and dose

- response relationship of endocrine disruption of estro gen, androgen, and thyroid in humans and wildlife .” Although the hormones produced and used by invertebrates are not directly analogous to those of vertebrates (e.g., estrogen, androgen, and thyroid), growth, reproduction, development, and other aspects o f invertebrate physiology and life cycle are known to be under
endocrine control. EDSTAC went on to note that “ chemicals that affect these vertebrate hormones may also affect invertebrate hormones resulting in altered reproduction, development, and growth

. ” Tier 2 tests were designed to be definitive tests which generate sufficient data to characterize the specific hazard of the substance and provide sufficient
information on dose

- response and adverse effects to permit risk decisions.

Please comment on t he rationale and purpose of the assay as part of the Tier 2 testing in the EDSP, as described in Sections 2. 1 and 2. 2 of the MTTT ISR.

2. Test methods and their associated endpoint(s) should be scientifically relevant to the

biological processes of interest and should be demonstrated to be responsive to the specific type of effect/toxicity of interest. Each species presents unique charac teristics from a biological perspective and allows for specialized endpoints to address a specific toxicological
mode of action.

Please comment on the biological and toxicological relevan ce of the assay in regards to the stated purpose of characterizing endocrine disruptors, as described in Section 2 of the
MTT T ISR.

3. The test protocol should be sufficiently detailed and should include a description of what is

measured and how it is measured. The selection of endpoints within the assay should be

re flective of the biological processes of interest and the endpoints should be intrinsically relevant and have established sensitivity. The test protocol should demonstrate the ability to measure the endpoints and provide adequate performance criteria for e valuation.

Please comment on the selection, optimization and demonstration of the assay endpoints,
as outlined in Sections 3 and 4 of the MTT T ISR

.

4. Demonstration of the test method performance should be based on the testing of reference

chemicals representative of the types of substances for which the test method will be used. Test substances should adequately represent an appropriate range of res ponses and physical/chemical properties for which the test method is proposed to be appropriate. The selection of the most appropriate statistical approaches depends in part on the nature of the data and also on the design of the validation study. Statis tical and non

- statistical methods used to analyze should be described.11 4.1 Please comment on the selection of test substances and methods (analytical and statistical where appropriate) chosen for the demonstration and validation of the MTT T

assay.

The proposed MTTT study design uses three replicates per control and treatment group and additional replicates require greater resources.

4.2 Please comment on the proposed level of replication and the subsequent statistical analysis of the data.

5. Consi dering the variability inherent in biological and chemical test methods, a test method needs to be repeatable and reproducible. A test is robust and reliable if the results are repeatable and reproducible within a laboratory and between different laborato ries, respectively. A test protocol should provide sufficient guidance to ensure proper and consistent performance
across labs and chemicals.

Please comment on the test method robustness and reliability and the repeatability and reproducibility of the results obtained with th e MTTT assay.

6. T he test protocol should be descriptive enough to be fully transferable to a functional

laboratory. The protocol should describe the methodology of the assay in a clear and concise manner so that a laboratory could comprehend the objective, conduct the ass ay, observe and measure prescribed endpoints, compile and prepare data for statistical analyses, and report the
results. Section 5 of the MTT T ISR outlines the process of and challenges experienced in the
inter

- laboratory studies.

6.1 Please comment on the transferability across labs and provide any suggestions or
recommendations for improvement of the MTTT assay

.

B ased on the validation results using the two different invertebrate species, mysid (MTTT) and copepod (HCDRT), the EPA is proposing the mysi d protocol as the preferred Tier 2 assay.

6.2 Please comment on the rationale to recommend the mysid protocol as the preferred Tier 2 invertebrate assay , as described in Section 6.4 of the MTTT ISR.

7. The purpose of the validation process is to determine the readiness of a test for inclusion in a

testing program. A component of readiness of a test is the evaluation of the usefulness and
limitations of the test , including the classes and types of tes t substances that can and cannot be
tested.

Please comment on the strengths and/or limitations of the assay , as described in Section 6
of the MTTT ISR

.12 8. There is sufficient evidence to indicate that EDCs can disrupt normal development and

reproductive success, however the sensitivity of the F 1 generation compared to the F 0 is less clearly defined.

Please comment from a scientific and risk assessment perspective on the value added of multiple generations in the MTTT assay